1,25 dihydroxyvitamin D (1,25(OH)2 D)-mediated intestinal calcium absorption is an important adaptive mechanism utilized by the body when calcium status is low. Low calcium absorption efficiency is a risk factor for osteoporosis and intestinal resistance to 1,25(OH)2 D action develops with aging and after menopause. Our long-term objective is to clarify the mechanisms used by 1,25(OH)2 D to promote calcium absorption and to determine how dysfunction in the regulatory control of intestinal calcium absorption during aging or due to menopause leads to reduced absorption efficiency and, ultimately, low bone density. Recent research demonstrates that 1,25(OH)2 D rapidly activates scr kinase, protein kinase C (PKC), and MAP kinases and that inhibition of these kinases blunts 1,25(OH)2 D-mediated activation of the CYP24 gene. The goal of the proposed research is to determine how the adaptive increase in intestinal calcium absorption due to 1,25(OH)2 D-dependent; vitamin D receptor (nVDR)-mediated gene activation is influenced by the basal or induced activity of these kinases. The specific aims of this project are to: (1) Identify the nVDR-mediated genomic pathways controlling intestinal calcium absorption that are modulated by 1,25(OH)2-induced activation of src kinase, PKC, and MAPK, (2) Establish the mechanisms by which 1,25(OH)2 D-induced kinase activation promotes 1,25(OH)2 D-mediated gene expression and intestinal calcium absorption, and (3) Identify new, 1,25(OH)2 D-regulated transcripts in differentiated enterocytes and determine whether their regulation is modulated by activation of protein kinases. We will accomplish these aims by studying the effect of 1,25(OH)2 D in a well-characterized cell culture model (Caco-2 cells) and in the small intestine of mice. Biological actions of 1,25(OH)2 D will be studied in the presence of activators and inhibitors of protein kinases (pharmacologic inhibitors, dominant negative kinases) and the rapid actions of vitamin D (vitamin D analogs), nVDR action and function will be studied with cellular imaging, RT-PCR, DNA microarrays, Western blotting and chromatin immunoprecipitation (CHIP) assays. Elucidating the mechanism for how signal transduction pathways influence the genomic actions of 1,25(OH)2 D will permit us to design prevention and pharmacologic intervention strategies to enhance intestinal calcium absorption, especially when vitamin D resistance associated with aging or estrogen deficiency is present.